1887
J. Org. Chem. 1993,58, 1887-1894
Diastereoselectivity (Enantioselectivity) of Aldol Condensations Catalyzed by Rabbit Muscle Aldolase at C-2 of RCHOHCHO if R Has an Appropriately Placed Negatively Charged Group' Watson J. Lees2and George M. Whitesides' Harvard University, Department of Chemistry, Cambridge, Massachusetts 02138 Received October 6, 1992
D-Fructose 1,6-bis(phosphate)aldolase from rabbit muscle (RAMA,E.C. 4.1.2.13) catalyzes the aldol condensation between dihydroxyacetone phosphate (DHAP) and various aldehydes; the products are ketosugars with 3(S),4(R)stereochemistry. When racemic a-hydroxy aldehydes are condensed with DHAP, two diastereomeric products are formed. This paper demonstrates that RAMA can kinetically resolve a-hydroxy aldehydes with a negative charge removed four or five atoms from the aldehydic center. Kinetic resolution of either uncharged a-hydroxy aldehydes, or of a-hydroxy aldehydeswith a negative charge removed three or seven atoms from the aldehydiccarbon,is generally not as successful.
Introduction The stereoselectiveformation of carbon-carbon bonds using aldol condensations is important in organic synthesis.3 We" and others8J' have used the enzyme fructose 1,6-bis(phosphate) aldolase (E.C. 4.1.2.13; from rabbit to synthesize muscle, rabbit muscle aldolase, RAMA)10-12 carbohydrates. In nature, RAMA catalyses the aldol interconversion between dihydroxyacetone l-phosphate (DHAP), glyceraldehyde 3-phosphate (G3P), and D-frUCtose 1,6-bis(phosphate). The product of a RAMA-catalyzed aldol condensationbetween DHAP and an aldehyde that is chiral at C-2 (C-5 in the adduct with DHAP) can, in principle, produce either of the two epimeric products correspondingto the two enantiomers of the aldehyde (eq 1)or a mixture of the two. The ratio of these two products Hf?z-k2
OP032. LHf:po32-
OH
-Jo
OH
-0
H
OH
R
('1
OH
R
R
depends upon three factors: the kinetic selectivity of the enzyme, the relative thermodynamic stability of the two (1) Supported by the NIH, Grant GM30367. (2) Natural Science and Engineering Research Council of Canada, Centennial Scholar. (3) AsymmetGSynthesis; Morrison,J. D.,Ed.; Academic: New York, 1983; Vole. 2 and 3. (4) Straub, A.; Effenberger, F.; Fischer, P. J. Org. Chem. 1990, 55, 3926-3932. 15) Borvsenko. C. W.: SDaltenetein. A.: Straub.. J. A.: Whitesides. G. M. J. Am: Chem: SOC.19s'S, 111,9275-9276. (6) Turner, N. J.; Whitesides, G. M. J. Am. Chem. SOC.1989, 111, 624-627. (7) Bernarski, M. D.;Simon, E. S.; Bischofberger, N.; Fessner, W.-D.; Kim, M.-J.;Lees, W. J.; Saito, T.; Waldmann, H.; Whitesides, G.M. J. Am. Chem. SOC.1989,111,627-635. (8)Scbultz, M.; Waldmann, H.; Vogt, W.; Kunz, H. TetrahedronLett. 1990,31,867-868. (9) Pederson, R. L.;Kim, M.-J.;Wong, C.-H. TetrahedronLett. 1988, 29, 4645-4648. (10) Horecker, B. L.; Tsolas, 0.;Lai, C. Y. Enzyme 1972,7,213-258. (11) The Enzyme Handbook; Barman, T. E., Ed.; Springer-Verlag: New York, 1969; Vol. 11, pp 736-737. (12) Willnow, P.Methods ofEnzymatic Analysis, 3rd ed.;Bergmeyer, H. U., Bergmeyer, J., Graesi, M., Eds.; Verlag Chemie: Weinheim, 1984; Vol. 11, p 146; VOl. IV,p 346-353.
OO22-3263/93/1958-1887$04.OO/O
products, and the extent to which the mixture of products approaches thermodynamic equilibrium. In this paper, we address the kinetic resolution of aldehydes (eq 1) in aldol condensations catalyzed by RAMA. The objective of this work is to establish the practicality of RAMA in setting the stereochemistryat C-5 (and thereby f i i three contiguous chiral centers) starting from a mixture of enantiomeric aldehydes and (achiral) DHAP.I3 For reactions involvingtwo enantiomersof the aldehyde, the kinetic selectivity of RAMA is given by the ratio of initial rate constants k d k z (eq 1). Lardy and co-workers have reported that RAMA shows a high kinetic selectivity for D- over L-glyceraldehyde3-phosphate (k1lk2 >> l).I4 In contrast, our preliminary survey established that RAMA showed no kinetic selectivity for D-glyceraldehyde over The contrast between these L-glyceraldehyde(k1lk2 = l).16 results suggested that a negative charge remote from the aldehyde group might be important in obtaining kinetic selectivity. Independent evidence has suggested that a negative charge can increase the rate of aldolase-catalyzed reactions: R u t t d 6 reported that both fructose 1,6-bie(phosphate) ( K m = 15 pM, Vm, = 6250) and sorbose 1,6bis(phosphate) ( K m = 44 pM, V m u = 360) are better substrates than their monophosphate analogues fructose 70) and sorbose l-phosphate ( K m = 6300 pM, Vm, l-phosphate ( K m = 600 pM, Vm, = 95). We have reported previously that aldose phosphates react more rapidly with DHAP than with the corresponding nonphosphorylatsd aldosein aldol condensationscatalyzedby RAMA.' These results suggest that an appropriately placed negative charge enhances the rate of RAMA-catalyzed aldol condensations. Barker" has reported that poly01 diphosphates-mimics of the product obtained by an aldol condensation between a negatively charged aldehyde and DHAP-are stronger inhibitors of RAMA than analogous monophosphates; specifically,arabitol, xylitol, and ribitol l,Bbis(phosphate) (Ki= 40-3 p M ) were better inhibitors (13) Sih, C. J.; Wu, S.-H. Top. Stereochem. 1989,19,63-125. (14) Tung, T.-C.; Ling, K.-H.; Byrne, W. L.; Lardy, H. A. Biochem. Biophys. Acta 1954,14,488-494. (15) Thediastereoselectivitywasdeterminedas deacribedinthegeneral procedure. The resulting spectra were also compared with fru1-phosphate and the product of the aldolase catalyzed condewtion between DHAP and t-glyceraldehyde. (16) Richards, 0.C.;Rutter, W. J. J. Bid. Chem. 1961,236,3185-3192. (17) Hartman, F. C.; Barker, R. Biochemistry 1966,4, 1068-1075. (0 1993 American
Chemical Society
Lees and Whitesides
1888 J, Org. Chem., VoI. 58,No.7, 1993
6.4A
4
+a 3 A + Figure 1. Important residues in the active site of RAMA and the proposed transition state for the enzymatic formation of fructose 1,6-bis(phosphate)from dihydroxyacetone phosphate (DHAP) and glyceraldehyde3-phosphate(G3P). The ionization states of the phosphates, arginine, and lysines in the active site are unknown. The G3P is presumed to be trans-extended in the transition state.
l.27In this transition state, glyceraldehyde 3-phosphate is trans-extended. When this transition state is viewed perpendicularly to the trans-extended glyceraldehyde 3-phoaphate, the C-1 of glyceraldehyde 3-phosphate overlaps with the C-3 of the eneamine formed between Lys-229 and the ketone of DHAP. Molecular modeling using the MM2 parameter set28showed that the in-plane distance between an oxygen on the 3-phosphate of glyceraldehyde 3-phosphate and the Nf of Lys-229 in the proposed transition state is 8.3 A. The distance between the IV of Lys-229 and Lys-107 in the unoccupied crystal structure (using data kindly supplied by Prof. Sygusch and co-workers) wae 8.9 A.24 The similarity of these two distances suggests that group-specific interactions (hydrogen bonds and/or charge-charge interactions) make important contributions to the binding energy of FDP to the enzyme and subsequent conversion to product. The unoccupied crystal structures of human muscle29 and drosophila m e l a n ~ g a s t ealdolase r~~ have also been solved and shown to be similar to that of RAMA. To investigate the hypothesis that a negative charge is important in obtaining kinetic selectivitywith RAMA, we examined the kinetic selectivity of a-hydroxy aldehydes of general structure R(CH2)nCHOHCH0, where R was CH3, OH, or CO2H and 0 < n < 6. We chose substrates containing an a-hydroxy group in order @ mimic the natural substrate. We chose a carboxyl group rather than other negatively charged groups (sulfate,sulfonate, phosphate, or phosphonate) because the carbonyl group was conveniently accessible by synthesis and because the carboxy-terminatedproducts from the aldol condensation are potentially useful in carbohydrate synthesis.
Results
Synthesis of Aldehydes. The aldehydic substrates than the corresponding l,4-anhydro 5-phosphates (Ki= (1-7, Table I) were usually prepared by the hydrolysis of 4000-1000 pM). These results suggest that an approprithe dialkyl acetal (see supplementary material for the synthesisof the dialkylacetals). To show that the product ately placed negative charge enhances the binding of of the hydrolysis was predominantly an aldehyde (in its substrate to RAMA. various forms: hydrate, aldehyde, dimer, and polymer),3l Several groups have reported that the binding of fructose the dimethyl acetals of the five- and six-carbon carboxy1,6-bis(phosphate) to RAMA involves important interterminated aldehydeswere regenerated in acidic methanol actionsbetween charges.1s24 The active site of the enzyme and compared to the original acetal. The five-carbon containsthree residuesthat have been proposed to interact carboxy-terminated aldehyde formed the dimethyl acetal with fructose 1,6-bis(phosphate): Lys-229 may form a of the methyl ester and the dimethyl acetal of the lactone Schiff's base with C-2,18J9and Arg-14a20and L y ~ - 1 0 7 ~ l - ~ ~ in acidic methanol. may interact with the 1-and 6-phosphate groups, respecAldol Condensations. The a-hydroxy aldehyde and tively. The crystal structure of RAMA has been resolved DHAP were mixed together in two NMR tubes (5Ck150 to 2.7 A24 and is consistent with this model (Figure 1). mM of each). One tube contained more aldehyde than The proposed transition state for the C-C bond forDHAP (approx. 3:2); the other tube contained less mation in the active site of RAMA is depicted in Figure aldehyde than DHAP (approx. 2:3). The initial concentration of DHAP was determined using an enzymatic (18) Lubini, D. G. E.; Christen, P. R o c . Natl. Acad. Sci. U.S.A. 1979, assay.12 The initial concentration of a-hydroxy aldehyde ~
~~
~~~
76,2527-2531. (19) Lai, C. Y.; Nakai, N.; Chang, D. Science 1974, 183, 1204-1206. (20) Lobb, R. R.; Stokes, A. M.; Hill, H. A. 0.;Riordan, J. F. FEES Lett. 1975, 54, 70-72. (21) Shapiro, S.; Enser, M.; Pugh, E.; Horecker, B. L. Arch. Eiochem. Eiophys. 1968, 128, 554-562. (22) Anai, M.; Lai, C. Y.; Horecker, B. L. Arch. Eiochem. Eiophys. 1973,156, 712-719. (23) Palczeweki, K.;Hargrave, P. A.; Folta, E. J.; Kochman, M. Eur. J . Eiochem. 1985,146,304-314. (24) Sygusch, J.; Beaudry,D.;Allaire,M.Proc. Natl. Acad. Sci. U.S.A. 1987,84,7846-7850. An updated version wan kindly supplied by Prof. Sygusch. (25) Ross, I. A,; O'Connell, E. L. J. Eiol. Chem. 1977,252, 479-482. (26) Biellmann, J. F.; OConnell, E. L.; Row, I. A. J. Am. Chem. Soc. 1969,91,6484-6488.
(27) The cleavage of the C-C bond or a conformation change in the enzyme during departure of glyceraldehyde-3-Pie believed to be the slow step in the breakdown of fructose 1,6-bis(phoaphate).%.' (28) Still, W. C.; Mohamadi, F.; Richards, N. G . J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T. MacroModel V2.0;Department of Chemistry, ColumbiaUniversity, New York, NY. (29) Gamblin, S. J.; Cooper, B.; Millar,J. R.; Davies, G . J.; Littlechild, J. A.; Watson, H. C. FEES Lett. 1990,262, 282-286. Rossi, F. A,; Struck-Donatz, M.; (30) Heater, G.; Brenner-Holzach,0.; Winterhalter, K.H.; Smit, J. D. G.; Piontek, K.FEES Lett. 1991,292, 237-242. (31) By 'H NMR spectroscopy it wan determined that the aldehyde exists an a 40-10076 monomer depending on the aldehyde.
J. Org. Chem., Vol. 58, No. 7, 1993 1889
Aldol Condensations Scheme I. Reaction Sequences Used To Determine the Absolute Stereochemistry of the Products OCHCHOH(CHI)nCOONa
16 (for n i l )
NaBH4
*
HOCH*CHOH(CH&COONI
17 (for 17-2)
1 I
18 (for n-3)
of known a h l u t e stereochemistryand o p t i d rotation.For 7-hydroxy-8-oxooctanoate(4, n = 5), the esterification product, methyl 7,8-dihydroxyoctanoate,was synthesized from 2,3-isopropylidene-~-glyceraldehyde.In all four cases, the lactone or ester and, by inference, the residual aldehyde had the Sconfiguration. We conclude,therefore, that RAMA reacts preferentidy with the aldehydehaving the R configuration. Calculation of Diastereoselectivity. The diastereoselectivityof the reaction between an aldehyde and DHAP was estimated by the integration of the 'H NMR spectra of the enzyme-catalyzedreaction mixtures. We calculated the enantiomericratio, E, using eq 2, where c is the percent
In [ I - c(1 + ee(P))l (kmtlKm)A= E In [I - ~ ( 1 ee(P))I (kmJKm),
10 (for n-5)
5
20
21
H
O
U
0
w
19 (for n-5)
was determined relative to the concentration of DHAP by integration of the 'H NMR spectrum. RAMA was then added to each of these NMR tubes, and the relative concentrationsof the monomericaldehyde,nonmonomeric aldehyde (dimeric and polymeric), and each of the two diastereomericproducts were determined as a function of time by integration of 'H NMR spectra taken at different times. We measured the relative concentrations of the various componentsin solution using two different procedures. In the first procedure, we were interested in the relative concentrations of monomeric aldehyde and each of the diastereomeric produds (vide infra). We integrated the resonances corresponding to the following: the a CHOH group of the monomeric aldehyde, the H-4 proton of the diastereomeric product having the fructose stereochemistry; and the H-4 and/or H-5 proton (in some cases the resonances overlapped) of the diastereomeric product having the sorbose stereochemistry. In the second procedure,we were interested in the s u m of the concentrations of aldehyde (monomer,dimer, and polymer) and produds relative to the concentration of products ([total aldehyde productsl/[productsl). The s u m of the concentration of total aldehyde and products ([total aldehyde + products]) was determined by integrating the 'H NMR resion of the following (1) a CH2 group a to a carboxylicacid (for carboxy-terminated aldehydes); (2) an alkyl CH3 group (foralkyl-terminated aldehydes);and (3) the (CH2Inchain (for hydroxy-terminated aldehydes). We determined the relative concentrations of the diastereomeric products as described in the first procedure. Determination of Absolute Stereochemistry. In the RAMA-catalyzed condensation of 3-hydroxy-4-oxobutanoate (1, n = 1, Scheme I), 4-hydroxy-5-oxopentanoate (2, n = 2), 5-hydroxy-6-oxohexanoate(3, n = 3), and 7-hydroxy-8-ox~ooctanoate(4, n = 5) with DHAP,the stereochemistryof the residual aldehydeat approximately 50% conversion was determined by reduction of the reaction mixture followed by lactonization (n = 1,2, or 3) or esterification (n= 5) and comparison with a compound
+
(2)
conversion divided by 100, ee(P) is the diastereomeric excess of the product, K m is the Michaelis constant, kat is the turnover number, and k,JK, is the specificity constant for enantiomer A or B.36 The value of E is the ratio of initial rates, kllkz in eq 1, in the presenceof racemic aldehyde; it is independent of the starting concentration of aldehyde or DHAP. Since the error in the calculation of the percent conversion is probably i5% absolute, the relative error in values of E is larger for large values of E. As a consequence, we report values of E greater than 10 as simply >lo. Values of E were calculated using two separate definitions of percent conversion: [products]/ [totalaldehyde + product], and [products]/ [monomeric aldehyde product]. The former calculationis valid when the rate of formation of monomeric aldehyde (hydrate) from oligomeric forms is fast relative to the rate of formation of product (eq 3, k~ > k,[DHAP]). The latter
+
monomeric aldehyde
k,
b
RAMA / DHAp
two
condensation products (3)
dimeridpolymeric aldehyde
calculation is valid when the rate of formation of monomeric aldehyde (hydrate) from oligomeric forms is slow relative to the rate of formation of product (eq 3, kd < k,[DHAPl), that is, in essence, when the nonmonomeric aldehydedoes not participate in the condensationreaction. The former calculation provides the minimum value of E and the latter calculation provides the maximum value of E. The value of E is therefore reported as a range spanning these two estimates (Table I). The discrepancybetween the two methodsof calculation will be larger at higher conversion. In order to minimize this discrepancy, the 'H NMR spectrum taken at approximately 50 9% conversion of monomeric aldehyde was (32) Taniguchi, M.; Koga, K.;Yamada, S. Tetrahedron 1974,30,36473562. Herdeis,C. Synthesis 1986,232-233. Ravid, U.; Silverstein, R. M.; Smith, L. R. Tetrahedron 1978,34, 1449-1452. Vigneron, J. P.; Meric, R.; Larcheveque, M.; Debal, A.; Lallemand, J. Y.; Kunewh, G.;Zageatti, P.; Gallois, M. Tetrahedron 1984,40,3621-3529. (33) Mori, K.;Takigawa, T.; Matsuo, T. Tetrahedron 1979,36,933940. Saito, S.; Hasegawa, T.; Inaba, M.; Nishida, R.; Fujii, T.;Nomizu, S.;Moriwake, T. Chem. Lett. 1984,1389-1392. Tanaka, A.; Yamashita, K.Synthesis 1987,670-573. (34) Gerth, D. B.;Geise, B. J . Org. Chem. 1986,51,3726-3729. Taylor, R. J. K.: Wieeins. K.: Robinson. D. H. Synthesis 1990. 589-690. Murahashi, S.rf;Naota; T.; Ito, K.i Maeda, Y:; Taki, H. J. Org. Chem. 1987,52,4319-4327. (36) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.;Sih, C. J. J. Am. Chem. SOC.1982, 104, 7294-7299.
1890 J. Org. Chem., Vol. 58, No. 7, 1993 Table I. Valuer of E for Variour a-Hydroxy Aldehydes (RCHOHCHO) hydroxy condeneation R aldehyde no. product no. Eo HOCH2 1 Na20sPOCHz 8 >10 NaOgCH2 1 9 3-4 Na02C(CHzh 2 10 >10 NaO&(CHd3 3 11 >10 NaOG(CHzh 4 12 4-6 HOCH2(CH2k 5 13 2-3 HOCHa(CHd3 6 14 2-3 H&(CHzh 7 15 1 ThoeevaluesofEgivenaarangesrepreeentdifferentassumptiom concerning the extent to which the aldehyde is in thermodynamic equilibrium with equivalent forma (hydrate,dimer,other)under the reaction conditions. See the test for diecumion.
Lees and Whitesidea
4-i
19
4
10
m
-%
*y.oOH
used in the calculations. In some cases with conversions lower than 50%, resonances corresponding to the minor product could not be detected. The values of E provided in Table I do not, in most cases,describethe diastereomeric excess in the usual form as a function of conversion from 0 to 100%. We are unable to report standard values of E because the aldehyde exists in various forms: The ee of the monomeric aldehyde (hydrate) in solution at a given conversion, therefore, may not be the same as the ee's of the aldehyde in its oligomeric forms. Nevertheless, these values of E provide useful qualitative descriptions of the kinetic selectivityof RAMA toward the aldehydesstudied in this work. Synthesis of SugarPhosphates. The aldehyde (2.53.0 equiv), DHAP (1 equiv, in most cases 2 mmol), and RAMA were stirred together until the reaction reached approximately80% conversion. At this point the reaction mixture was purified by ion-exchange chromatography. For 4-hydroxy-5-oxopentanoate(2) and 5-hydroxy-6oxohexanoate (31,the ratio of the fructoseproduct (reaction with R-aldehyde) to sorbose product (reaction with S-aldehyde)in the isolated material was greater than 101; this result confirmed the inferences from the NMR experiments. For 3-hydroxy-4-oxobutanoate(1) and 7-hydroxy-&oxooctanoate (4) the ratios of the fructose product to sorbose product in the isolated material were 3:l and 7:1, respectively, again confirming the inferences from the NMR experiments.
Discussion RAMA shows high diastereoselectivity-as measured by E-between enantiomeric a-hydroxy aldehydes with negative charges five or six bonds removed from the aldehydic carbon. RAMA exhibits lower diastereoselectivity between a-hydroxy aldehydeswith negative charges four or eight atoms removed from the aldehydic center and between a-hydroxyaldehydea with no negative charge. These results are tabulated in Table I. Analysis of the crystal structure of RAMA" helps to rationalize these observations. As discussed above, the active site of RAMA contains a number of charged residues that may isteract with the ligands*a23(Figure 1). We believe that it is important that the product of the aldolcondensationspans the distance between the lysine groups in order to obtain high diastereoselectivity. This conformation is more easily established when a charged group in the aldehyde can interact with Lys-107. In order to make comparisons between the transitionstate structures formed from a series of aldehydes, we
0
k R - l
Figure 2. Stereoselectivity of the a l d o h - c a t a l y d reaction (E)plotted againstthe C-4 to terminal oxygendistance of varioue trans-extendedaldol condeneationproducta (8-12) aa determined by force-field calculations.
R-ddcbyde
&aldehyde
Nu = enzyme bound D W
Figure 3. End-on view of the attack of DHAP (Nu) on each of the enantiomers of the aldehyde.
compared the distance between C-4 and the furtheat inplane oxygenof the trans-extended products from an aldol condensation using force field calculations (Figure2). We chose C-4 because this is the first carbon atom derived from the aldehydein the chain of the condensationproduct The distance between C-4 and an oxygen on the carbosylate of 6-deoxy-6-carboxyfructosel-phosphate (9; the product of the aldol condensation between DHAP and 3-hydroxy-4-oxobutanoicacid, 1) is 4.9 A. Thia distance is 1.5 A shorter than the distance between C-4 and an oxygen on the 6-phosphate of trans extended fructuae 1,6bistphosphate) (8). This conformationof FDP spans (vide supra) the distance between the two lysine residues in the unoccupied active site; consequently, compound 9 maybe too short to have an effective interaction between its terminal carbonyl group and Lys-107. The observation that RAMA shows low kinetic selectivity in the formation of this product (9) but high kinetic selectivity in the formation of compounds 10 (6.2 A), 11 (6.4 A), and 12 (7.5 A) is consistent with the hypothesis that formation of an ion pair is necessary for high kinetic selectivity. We hypothesize that the orientation and the location of the carbonyl group of the aldehyde are fixed in the active site of the enzyme. The two-point binding of the carbonyl group and the carboxylate in the five- and six-carbon carboxy-terminated aldehydes probably limits the rotational freedom of the a-hydroxy group in the bound aldehyde. This limited rotation causea the enzyme-bound form of one enantiomer of the aldehyde to present the a-alkyl chain or the a-hydroxy group on its si face; the other enantiomer of the aldehyde presents the sterically smaller a-hydrogen on its si face. In Figure 3, we assume that the aldehydic oxygen and the alkyl group are syn since this orientation will maximize the distance between the C-1phosphate and the terminal group. Thisconstraint
Aldol Condensations
restricts the transition state for each enantiomer to that shown in Figure 3. DHAP always attacks the si face of the aldehyde; any group blocking this attack should slow the rate of the catalyticreaction. Theseconstraintspredict that the R enantiomer of the aldehyde (the faster reacting enantiomer) presents a hydrogen on ita si face while t h e S enantiomer presents a bulkier group on its si face. Diastereoselectivity results from this difference. T h e eight-carbon carboxy-terminated aldehyde 4 may
or may not be able to form an ion pair with the lysine residue. If 4 forms an ion pair with the lysine residue, its aUtyl chain should still be flexible because the distance between C-1 and t h e terminal oxygen is 3.6 A greater than t h e corresponding distancein fructose 1,6-bis(phosphate). This added flexibility should allow rotation about the a-hydroxy carbon, in a fashion s i m i i to noncharged aldehydes, removing the constraint that the aldehyde and alkyl group must be syn to each other. If 4 does not form an ion pair then the constraint t h a t the molecule is fiied at two points is removed. This added flexibility or loss of the ion pair may explain the lower kinetic selectivity of t h e enzyme toward 4 relative to the other charged aldehydes (2,3). We conclude that RAMA can provide diastereoselectivity in the reaction of DHAP with a-hydroxy aldehydes, provided that the aldehydes possess a negatively charged atom removed at least five bonds from the aldehydic carbon (Table I). Analysis of the unoccupied crystal structure suggests that an ion pair formed between the substrate and Lys-107is required to produce diastereoselectivity. If this ion pair cannot be formed-either because there is no negative charge on the substrate or because the substrate is unable to reach Lye-107 with its negative charge-then little or no diastereoselection occurs.
Experimental Section Materials and Methods. Chemicals were purchased from Aldrich and were reagent grade. Enzymes and biochemicalswere obtained from Sigma. Optical rotations were measured with a Perkin-Elmer 241 polarimeter. NMR spectrawere recordedusing tetramethylsilane (TMS) or chloroform (CHCb) as an internal standard or dioxane as an external standard. Condensation Reaction. General Procedure. A freshly prepared solution of DHAP (pH 7.0) in D20 (0.5 mL) was added to a solution of aldehyde in DzO (pH 7.0,0.5 mL). The relative concentration of these two components was determined by integration of the 'H NMR spectrum. Two 5-mm NMR tubes were prepared: one containing a slight excess of aldehyde, the other one containing a slight excess of DHAP. A lH NMR spectrum was then acquired (t = 0). Aldolase was added, and the reactions were followed by lH NMR analysis. Condensation Reaction for 4-Hydroxy-5-oxopentanoic Acid (2). The relative concentration of the two stock solutions (DHAP and the lactone of 4-hydroxy-5-oxopentanoicacid) were determined as in the general procedure. A solution containing a slightexof aldehyde relative to DHAP was made up. Sodium deuteride was added slowly until the solution maintained a pH value of 12 (opening of the lactone). 4-Hydroxy-5-oxopentanoic acid (resonances attributed to hydrate): lH NMR (400 MHz, D2O) 6 4.59 (d, J = 4.9 Hz,1H), 3.22 (ddd, J 9.4,4.9,3.4 Hz, 1H), 2.10 (ddd, J = 15.0,9.6,5.9 Hz, 1H), 2.00 (ddd, J = 15.0, 9.2,6.7 Hz,1H), 1.61 (dddd, J = 14.0,9.8,6.7,3.3 Hz, 1H), 1.39 (dtd, J 14.3, 9.3, 5.9 Hz, 1 H); 'W NMR (100 MHz, D20) 6 182.94,91.56,73.39,33.53,27.91; HRMS-FAB [M - HI- calcd for C&Od 131.0344, obsd 131.0346. The pH was adjusted to pH 7.0 with DC1. The solution was then placed in two 5-mm NMR tubes (0.9 mL). One of the tubes was diluted with the stock DHAP solution (0.1 mL, excese DHAP tube), the other tube was diluted with D2O (excess aldehyde tube). A lH NMR spectrum
J. Org. Chem., Vol. 58, No.7, 1993 1891 was then acquired ( t = 0) of each tube. Aldolase was added, and the reactions were followed by 'H NMR analysis. 6-Carboxy-6-deoxyfructore1-Phosphate (9) and 6-Carboxy-6-deo.yrorboae 1Phosphnte. Sodium 3-hydroxy-doxobutanoate diethyl acetal (1.31 g, 5.7 mmol), water (30 mL), and ion-exchangeresin (Dowex 5OW-X8,H+form, 13.9 g) were stirred together for 16 h. The reaction mixture was filtered, p d d y concentrated at 1Torr, and adjusted to pH 7 with 1N NaOH. To this solution was added a solution of DHAP (20 mL of a 100 mM solution,2 "01). The pH was readjusted to pH 7. Aldolase (50 U)was added and the reaction followed by monitoring the consumption of DHAP. After 2.5 h (80% completion), the reaction mixture was purified by anion exchangechromatography (AGl-X8 bicarbonate form/eluant: triethylammonium bicarl-phoebonate 0-600 mM) to provide 6-deoxy-6-carboxyf~ctc8e phate and 6-deoxy-6-carboxysorbe 1-phosphate as their triethylammonium salts. Due to a contamination with 10 mol % of the starting aldehyde, the sample was repurified by ionexchange chromatography on the above column. The triethylammonium salt solution was concentrated at l Torr to dryneas, dieeolved in water (100 mL), aciWied with ion-exchange resin (Dowex SOW-X8, H+ form), filtered, and adjusted to pH 7 with 1 N NaOH to form the sodium salt. The sodium salt solution was lyophilized to provide a mixture of 6-deoxy-6-carboxyfructoee 1-phosphateand 6-deoxy-6-carboxysorb 1-phosphate(388mg, 3 1 ratio) each of which exists as a mixture of two anomers (approximateratio 3 1 for each). The resonances corresponding to 6-deoxy-6-carboxyfructosa1-phosphate are labeled 'f", and the reaonancea comqxudiqto 6deoxy-&-carboxymrboee l-phoephate are labeled 'a". The resonances corresponding to the minor anomers are labeled "a" and the resonances correspondingto the major anomers are labeled 'b". Not all the resonancea corresponding to the minor anomera are reported: lH NMR (DsO, 500 MHz) 6 4.40 (m, 5-1, 4.36 (dt, J = 7.9, 5.9 Hz, 1H, Seb), 4.09-4.05 (m, 4sb, Sfa), 4.03 (dd, J = 4.8, 2.3 Hz, 4aa), 3.94 (d, J = 2.0 Hz, 3ea), 3.91 (d, J = 8.0 Hz, 1H, 3fb), 3.88 (d, J = 5.0 Hz, 1H, 3sb), 3.86-3.81 (m, 5fb, 3fa), 3.80 (t,J = 7.8 Hz, 4fb), 3.71 (t,J = 5.5 Hz, 4fa), 3.68-3.53 (m,If,le), 2.43 (dd, J = 15.3, 4.3 Hz,6fbb3,2.39 (dd, J = 15.4,5.2 Hz, 6fa'), 2.30 (dd, J = 15.1,
6.0Hz,6sb'),2.32-2.27(m,6fa"),2.28(dd,J=15.6,8.4Hz,6fb"), 2.20 (dd, J = 15.3, 8.0 Hz, 6sb"). The resonances are labeled large (1, probably the major anomer of 6-deo.y-6-carboxyf~ctc8e 1-phosphate),medium (m,probably the minor anomer of 6-deo.y6-carboxyfructae 1-phosphate,or the major anomer of 6-deo.y6-carboxyeorboee1-phosphate),or small (8, probably the minor anomer of 6-deoxy-6-carboxyfructose1-phosphate), depending ontheir intensities: lacNMR (D20,100MHz) 179.37m, 179.151, 104.16m (d, J = 7.7 Hz), 100.94m (d, J = 8.5 Hz), 100.601 (d, J = 8.7 Hz),81.71m, 79.90s, 79.4Om,77.631,77.07m, 76.08m, 75.9Om, 75.681,66.48m (d, J = 5.9 Hz), 65.781 (d, J = 4.2 Hz), 65.02m (d, J = 4.3 Hz), 42.331,41.35m, 38.43s,37.63m [Some of the signals in the lBC NMR spectrum were assigned after the pH had been adjusted to reduce signal overlap]; HRMS-FAB [M- HI- calcd for C,HlaOloP 287.0168, obsd 287.0149. 6-Deoxy-6-methylenecarboxyfructoae 1-Phosphate (10) and 6-Deoxy-6-methylenecarboxysorboae IPhosphata 4-Hydroxy-5-oxopentanoicacid lactone (0.736 g, 6.4 m o l ) in water (20 mL) was stirred for 90 min while the solution was slowly evaporated at aspirator pressure. Monosodium phosphate (68 mg, 0.5 "01) was added, and the solution was maintained between pH 10.5 and 9.0 with 1N NaOH (6.0 mL). The solution was then adjusted to pH 7.0 with 1N HC1. To this solution was added a solution of DHAP (sodium salt, 20 mL of a 100 mM solution, 2 "01) at pH 7.0. Aldolase (25 U)was added and the reaction followed by monitoringthe consumptionof DHAP.After 2.5 h (85% completion), the reaction mixture was purified by anion-exchange chromatography (AGl-X8 bicarbonate form/eluant: triethylammonium bicarbonate o-600 mM) to provide 6 - d e o x y - 6 m e t h y l e n e c a r b o x ~1-phosphate ~~ as the triethylammonium salt. The triethylammonium salt solution was concentrated at 1Torr to dryness, dissolved in water (100 mL),acidifiedwithion-exchangeresin (DowexSOW-XB,H+form), fitered, and adjusted to pH 7 with 1N NaOHto form the sodium salt. The sodium salt solution was lyophilized to provide a mixture of two components: sodium 6-deoxy-6-methyleneboxyfructmee 1-phosphate (713 mg) which exists as a mixture of
1892 J. Org. Chem., Vol. 58, No. 7, 1993 two anomera (ratio 4:l) and sodium 6-deoxy-6-methylenecarboxysorboae l-phosphate (ratio of diastereomers (14:l)). The minor anomer of sodium 6-deoxy-6-methylenecarboxyfruche l-phosphate is labeled "a" and the major anomer is labeled "b": 'H NMR (DzO, 500 MHz) 6 3.89 (d, J = 8.2 Hz, 3b), 3.81 (d, J = 4.4 Hz, 3a), 3.76 (t,J = 7.9 Hz, 4b), 3.76-3.72 (m, 5a), 3.67 (dd, J = 10.5, 8.7 Hz, la'), 3.67-3.65 (m, 4a), 3.61-3.51 (m, la", 5b,
lb),2.11(ddd,J=14.9,10.1,6.0Hz,7a',7b'),2.03(ddd,J=14.9, 10.1,5.9 Hz, 7a", 7b"),1.97-1.61 (m, 6a, 6b); 13CNMR (Dz0,100 MHz) 182.54a, 182.37b, 104.15a (d, J = 6.7 Hz), 100.54b (d, J = 8.4 Hz), 81.91a, 81.51a, 79.66b, 79.42a, 77.59b, 75.90b, 65.7713 (d, J =4.2 Hz), 64.81a (d, J =4.4 Hz), 33.34a, 33,15b, 30.62b, 29.3% HRMS-FAB [M.- Hl- calcd for CeHl~Old>301.0324, obad 301.0318. The fractions from the AGl-X8 ion-exchange column containing aldehyde were combined, and 1.8mmol of DHAP and 950 units of RAMA were added. After 16 h, the reaction mixture was purified by chromatography as above to provide 606 mg of a mixture of sodium 6 - d e o x y - 6 - m e t h y l e n e c a r b o ~l-phos~ phate and sodium 6-deoxy-6-methyleneboxysorbosel-phosphate (ratio 1:1.2). After subtracting the NMR spectrum of sodium 6-deoxy-6-methylenecarboxyfructose l-phosphate from the NMR spectrum of the mixture, the resonancescorresponding to sodium 6-deoxy-6-methylenecarboxysorbose l-phosphate were determined. The minor anomer of sodium 6-deoxy-6-methylenecarboxysorbose l-phosphate is labeled "a" and the major is labeled "b" (ratio 1:3): lH NMR (DzO, 500 MHz) 6 4.02-3.94 (m, 4b, 5b, 3a, 4a, 5a), 3.90 (d, J = 3.7 Hz, 3b), 3.77 (dd, J = 11.8, 8.0 Hz, la'), 3.61 (d, J = 6.0 Hz, lb'), 3.61 (d, J = 5.9 Hz, lb"), 3.55 (dd, J = 11.7, 7.0 Hz, la"), 2.15-2.00 (m, 7a, 7b), 1.77-1.61 (m, 6a, 6b'), 1.58-1.51 (m, 6b"); l*CNMR (DzO, 100MHz) 182.39 (a b), 105.29a (d, J = 7.3 Hz), 101.29b (d, J = 8.6 Hz), 82.35a, 79.74a, 78.46b, 77.27b, 76.0313, 75.40a, 66.72b (d, J = 4.6 Hz), 64.65a ( d , J = 4.7 Hz), 33.66a, 33.47b, 26.33a, 25.23b; HRMSFAB [M - HI- calcd for C~lsOlOp301.0324, obsd 301.0353; [M - 2H + Nal- calcd 323.0144, obsd 323.0157. 6-Deoxy-6-ethylenecarboxyfructose l-Phosphate (11). Methyl 5-hydroxy-6-oxohexanoatedimethyl acetal (1.18 g, 5.7 mmol), water (30 mL), and 9 mL of 1 N NaOH were mixed together. After being stirred for 30 min, the reaction mixture was acidified with ion-exchange resin (Dowex 50W-X8, H+ form, 13.2 9). After being stirred for 18 h, the reaction mixture was filtered, partially concentrated at 1Torr, and adjusted to pH 7 with 1N NaOH. To this solution was added a solution of DHAP (20 mL of a 100 mM solution, 2 mmol). The pH was readjusted to pH 7. Aldolase (25 U)was added and the reaction followed by monitoring the consumpton of DHAP. After 2.5 h (80% completion),the reaction mixture was purified by anion-exchange chromatography (AGl-X8 bicarbonate form/eluant: triethylammonium bicarbonate (0-500 mM) to provide 6-deoxy-6ethylenecarboxyfructose l-phosphate as the triethylammonium salt. The triethylammonium salt solution was concentrated at 1 Torr to dryness, dissolved in water (100 mL), acidified with ion-exchange resin (Dowex 50W-X8, H+ form), filtered, and adjusted to pH 7 with 1N NaOH to form the sodium salt. The sodium salt solution was lyophilized to provide sodium 6-deoxy6-ethylenecarboxyfructose 1-phosphate (789 mg) which exists as a mixture of two anomera (ratio 3:l). Due to a contamination with 10mol % of the starting aldehyde, the sample was repurified by ion-exchange chromatography on the above column to yield 661 mg of product. The minor anomer is labeled "a" and the major anomer is labeled "b": 'H NMR (DzO, 500 MHz) 6 3.88 (d, J = 8.5 Hz, 3b), 3.80 (d, J = 4.0 Hz, 3a), 3.77-3.74 (m, 5a), 3.74 (t, J = 8.0 Hz, 4b), 3.68 (dd, J = 11.6,8.4 Hz, la'), 3.64 (dd, J = 5.6, 4.4 Hz, 4a), 3.59-3.52 (m, la"), 3.57-3.48 (m, 5b, Ib), 2.02-1.96 (m, 8a, 8b), 1.50-1.36 (m, 7a, 7b, 6a, 6b); 13C NMR (D~0,100MHz)183.22 (aand b), 104.15a (d, J =7.1 Hz), 100.49b (d, J = 8.4 Hz), 81.88a, 81.77a, 79.83b, 79.63a, 77.73b, 75.94b, 65.86b (d, J = 4.3 Hz), 64.81a (d, J = 4.3 Hz), 37.20b, 37.10a, 33.58b, 32.32a, 21.91a, 21.60b; HRMS-FAB [M - Hl- calcd for CgH1701J' 315.0481, abed 315.0486. 6-Deoxy-6-butylenecarboxyfructoeel-Phorphete (12). Methyl 7-hydroxy-8-oxohexanoate dimethyl acetal (0.713 g, 3.0 mmol), water (15 mL), and 4 mL of 1 N NaOH were mixed together. After being stirred for 2.5 h, the reaction mixture was lyophilized and dissolved in 300 mL water. The solution was
+
Lees and Whitesides acidified with ion-exchange resin (Dowex 50W-X8, H+ form, 14 9). After being stirred for 48 h, the reaction mixture was neutralized with 1N NaOH, filtered, and partially concentrated at 1Torr to a total volume of about 30 mL. To this solution was added asolution of DHAP (10 mL of a 100mM solution, 1mmol). The pH was readjusted to pH 7. Aldolase (100 U)was added and the reaction followed by monitoring the consumption of DHAP. After 1.0 h (85% completion), the reaction mixture was purified by anion-exchange chromatography (AG1-X8 bicarbonate fomdeluant: triethylammonium bicarbonate O-600 mM) to provide 6-deoxy-6-butylenecarboxyfructose l-phosphate as the triethylammonium salt. The triethylammonium salt solution was concentrated at 1 Torr to dryness, dissolved in water (100 mL), acidifiedwithion-exchange resin (Dowex 50W-X8,H+form), filtered, and adjusted to pH 7 with 1N NaOH to form the sodium salt. The sodium salt solution was lyophilized to provide a mixture of sodium 6-deoxy-6-ethylenecarboxyfructoeel-phosphate which exists as a mixture of two anomers (ratio 3:l) and sodium 6-deoxy-6-ethylenecarboxysorboee l-phosphate (ratio 7:l fructoee/sorboee). Due to a contamination with 10 mol % of the starting aldehyde, the sample was repurified by ion-exchange chromatography on the above column to yield 350 mg of product. The minor anomer is labeled "ar and the major anomer is labeled "b": 'H NMR (DzO, 500 MHz) 6 3.87 (d, J = 8.2 Hz, 3b), 3.81
(d,J=4.0Hz,3a),3.77-3.73(m,5a),3.73(t,J=8.1Hz,4b),3.69 (dd,J=11.4,7.7Hz,laf),3.63(dd,J=6.0,4.2Hz,4a),3.5~3.48 (m, la", 5b, lb), 1.96 (t, J = 7.5 Hz, loa, lob), 1.51-1.36 (m, 6a, 6b), 1.34 (pentet, J =7.4Hz,9a, 9b), 1.28-1.06 (m,8a, 8b,7a, 7b); 13C N M R (D20,100 MHz) 183.94 (a and b), 104.18a (d, J = 7.2 Hz), 100.46b (d,J =8.7 Hz),82.14a,81.89a,80.01b,79.79a, 77.82b, 76.08b, 65.9113 (d, J = 4.6 Hz), 64.84a (d, J = 5.4 Hz), 37.36 (a and b), 33.6513, 32.44a, 28.54 (a and b), 25.57 (a and b), 24.55a, 24.18b; HRMS-FAB [M - Hl- calcd for CllH22010P 343.0794, obsd 343.0812. The fractions from the second AGl-X8 ionexchange column containing aldehyde were combined, and 0.4 mmol of DHAP and 400 units of RAMA were added. After 24 h, the reaction mixture was purified by chromatography as above to provide a mixture of sodium 6-deoxy-6-butylenecarboxyfructose l-phosphate, sodium 6-deoxy-6-butylenecarboxysorbose l-phosphate (ratio l:l), and citrate (buffer salt from the added aldolase). Resonances corresponding to the minor anomer and major anomer of sodium 6-deoxy-6-butylenecaboxyeorbose l-phosphate are labeled "sa" and "ab", respectively. Resonances corresponding to the anomers of sodium 6-deoxy-6-butylenecarboxyfruche l-phosphate are labeled "f". Resonances corresponding to citrate are labeled "c": lH NMR (D20,500 MHz) 6 4.05-3.95 (m, h b , 3sa, 4sa, 5sa), 3.94 (dd, J = 4.7,3.4 Hz, 4sb), 3.88 (d, J = 3.5 Hz, 3sb), 3.77 (dd, J = 11.4, 8.7 Hz, lsa'), 3.59 (d, J = 6.4 Hz, lsb'), 3.59 (d, J = 6.3 Hz, lab"), 1.95 (t, J = 7.5 Hz, loa), 1.50-1.10 (m, 9s,8s, 78,681; I3C NMR (DzO, 100 MHz) 184.02 (sa, sb,f), 181.76 (c), 179.05 (c), 101.45 (d, J =7.7 Hz, ab), 82.89 (sa), 82.18 (f),81.94 (f), 80.04 (f),79.87 (f),79.75 (sa), 79.04 (ab), 77.84 (sb), 76.37 (ab), 76.09 (f), 75.55 (c), 75.11 (sa), 66.85 (d, J = 4.7 Hz, ab), 65.89 (f),45.51 (c), 37.44 (sa, sb, f),33.71 (f), 32.49 (0, 29.32 (sa), 28.60 (sa, ab, f), 27.96 (sb), 25.65 (sa, sb, f), 24.98 (sa), 24.81 (sb), 24.60 (f), 24.22 (f); HRMS-FAB [M - HIcalcd for CllH21013 343.0794, obsd 343.0812. 6-Deoxy-6-ethylfructose l-Phosphate (16) and 6-Deoxy6sthylsorbose l-Phosphate. After a mixture of 2-hydroxypentanal dimethyl acetal (560 mg, 3.78 mmol), ion-exchangeresin (Dowex 50W X-8H+form, 4.4 g), and water (10 mL)was stirred for 14 h, the mixture was filtered. The solution was mixed with dihydroxyacetone phosphate (20 mL of a 100 mM solution, 2 mmol), neutralized to pH 7.0 with 1N NaOH, and diluted to 100 mL. RAMA (100 U) was then added to the mixture. After 20 h (100%completion),the reaction mixture was purified by anionexchange chromatography (AGl-X8 bicarbonate form/eluant: triethylammonium bicarbonate 0-300 mM) to provide a mixture of 6-deoxy-6-ethylfructose l-phosphate and 6-deoxy-6-ethylsorbose l-phosphate as their triethylammonium salts. The triethylammonium salt solution was concentrated at 1Torr to dryness, dissolved in water (100 mL), acidifed with ion-exchange resin (Dowex 50 W-X8, H+ form), fitered, and adjusted to pH 7 with 1 N NaOH to form the sodium salt. The sodium salt solution was lyophilized to provide 587 mg of a mixture of 6-deoxy-6ethylfructose 1-phosphate and 6-deoxy-6-ethylaorbose l-phos-
Aldol Condensations phate (1:l ratio). The resonances corresponding to 6-deoxy-6carboxyfructoae 1-phosphate are labeled 'f", and the resonances corresponding to 6-deoxy-6-carboxysorbose1-phosphate are labeled 's". The resonances corresponding to the major anomers are labeled 'a", and the resonances corresponding to the minor anomers are labeled 'b". Not all the resonances corresponding to the minor anomers are reported 'H NMR (DzO, 500 MHz) 6 4.06-4.02 (m, 5sb), 4.00 (dt, J = 7.5,5.4 Hz, 5m), 3.95-3.92 (m, 4sa, 3sb, 4sb), 3.87 (d, J = 3.9 Hz, 3ea), 3.87 (d, J = 6.0 Hz, 3fa), 3.80 (d, J = 4.2 Hz, 3fb), 3.76 (q, J = 6.5 Hz, 5fb), 3.71 (t, J = 8.1 Hz, 4fa), 3.67 (dd, J = 11.5,7.8 Hz, lfb'), 3.63 (dd, J = 6.1, 4.4 Hz, 4fb), 3.59-3.51 (m, 5fa, lfa, lea, lfb", lsb"), 1.46-1.08 (m, 6f, 6s, 7f, 7s), 0.72-0.66 (m, %a, Sf). The resonances are labeled large (1, probably the major anomer of 6-deoxy-6-ethylfrude I-phosphate or 6-deoxy-6-ethyleorbe 1-phosphate) or small (e, probably the minor anomer of 6-deoxy-6-ethylfructoae l-phosphate or 6-deoxy-6-ethylsorh 1-phosphate),dependingon their intensities: 'BC NMR (D20,100 MHz) 101.391 (d, J = 7.6 Hz), 100.471(d,J=6.1 Hz),82.50s,81.79s,79.631,78.681,77.801,77.681, 76.271,76.121,75.47s,66.781,65.941,64.69s,64.38~,35.971,34.69s,
31.37~,30.231,18.60s,18.451,18.20s,17.921,13.231,13.191;HRMSFAB [M - HI- calcd for C&O ,&' 271.0583, obsd 271.0591. 6-Deaxy-6-ethylPructose and 6-Deoxy-6-ethylmrb. Water (200mL),acidphosphataae (200U), andamixtureof6-deoxy6-ethylfructoae 1-phosphate (15) and 6-deoxy-6-ethylsorbose 1-phosphate (1:l ratio, sodium salt, 491 mg)were mixed together and left at room temperature. After 48 h, the solution was concentrated in vacuo and purified by silica gel chromatography (eluent: acetone/CHzClz (1:5 going to 1:l))to provide 138 mg of a mixture of 6-deoxy-6-ethylfructmeand 6-deoxy-6-ethyhrbose. The mixture was purified by silica gel chromatography(methanov CHzCl2 (1:lO going to 1:5)). The early fractions were used to characterize 6-deoxy-6-ethylsorbose,and the later fractions were used to characterize 6-deoxy-6-ethylfructe. 6-Deoxy-6-ethylsorbose: The resonances corresponding to the major anomers are labeled 'a", and the resonances corresponding to the minor anomers are labeled 'b" (ratio 51): 'H NMR (D20, 500 MHz) b 4.04-3.92 (m, 3b, 4b, 5b), 4.00 (dt, J = 7.6, 5.2 Hz, 5a), 3.96 (dd,J=4.8,3.7Hz,4a),3.84(d,J=3.6Hz,3a),3.48(d,J=11.9 Hz, lb'), 3.38 (d, J = 12.0 Hz, la', lb"), 3.32 (d, J = 11.9 Hz, la"), 1.43-1.06 (m, 6,7), 0.73-0.70 (m, 8b),0.70 (t,J = 7.3 Hz, 8a). The resonances are labeled large (1, probably the major anomer of the compound) or small (8, probably the minor anomer of the compound), depending on their intensities. NMR of mixture (Dz0, 100 MHz) 105.118, 101.911, 81.958, 79.848, 78.611, 76.721, 76.261, 76.02s,63.601, 62.32~,31.43s,30.321, 18.598, 18.431, 13.17 ( 8 +I); HRMS-FAB [M + Na]+ calcd for CsH160s215.0895, obsd 215.0884. 6-Deoxy-6-ethylfructose:The resonancescorresponding to the major anomers are labeled -arr, and the resonances corresponding to the minor anomers are labeled 'b" (ratio 41): 1H NMR (D20, 400 MHz) b 3.84 (d, J = 8.3 Hz, 3a), 4.83-4.82 (m, 3b), 3.75 (t,J = 7.8 Hz, 4a), 3.75-3.72 (m, 5b), 3.58 (dd, J = 7.0, 5.2 Hz, 4b), 3.51 (td,J = 7.6, 5.2 Hz, 5a), 3.39 (8, lb), 3.35 (d, J = 12.0 Hz, la'), 3.28 (d, J = 12.0 Hz, la"), 1.46-1.35 (m, 61, 1.27-1.10 (m, 7),0.70 (t,J = 7.4Hz, 8). TheI3C NMRresonances corresponding to the second compound were determined after the resonances corresponding to the first compound had been subtracted from the 13C NMR spectrum of the mixture. The resonances are labeled large (1, probably the major anomer of the second compound) or small (8, probably the minor anomer of the second compound), depending on their intensities: 13CNMR of 80.40s, mixture (DzO, 100 MHz), 103.82~,101.89,100.931,82.15s, 79.801, 78.60, 77.941, 76.69, 76.23, 75.181, 63.58, 62.698, 62.601, 62.29,35.941,34.4Os,31.42,30.31,18.59,18.42,18.1Os, 17.891,13.14 (a 1)ppm; HRMS-FAB [M + Nal+calcd for CeHleOs 215.0895, obsd 215.0898. 3-Hydroxybutyrolactone (16). The reaction of sodium 3-hydroxy-4-c\xobutanoate (1, 1.74 mmol) with DHAP in D2O (about 20 mL) was run as described in the condensation procedure. At 40% conversion the reaction was quenched with sodium borohydride (305 mg, 8.06 mmol). The reaction mixture was acidified to pH 1.0 with 1N HC1, concentrated in vacuo to 4 mL, and extracted with ethyl acetate (2 X 100mL). The ethyl acetate solution was then dried (MgSO,) and concentrated at aspirator pressure. The residue was purified by silica gel chromatography (ethyl acetate/hexane (1:1-51)) to give 3-hydroxybutyrolactone
+
J. Org. Chem., Vol. 58, NO.7, 1993 1893 (16,24 mg, 0.24 mmol): lH NMR (CDCl3,400 MHz) b 4.65 (ddt, J = 6.0,4.4, 1.7 Hz, 1 H), 4.39 (dd, J = 10.3, 4.4 Hz, 1H), 4.28
(dt, J = 10.3, 1.3 Hz, 1 H), 3.25 (br s, 1 H), 2.72 (dd, J = 18.0, 6.0 Hz, 1H), 2.49 (dt, J = 18.0, 1.4 Hz, 1H); lacNMR (CDCg, 100MHz) 176.73,76.18,67.40,37.75ppm; HRMSCI [M N&1+ calcd for CtHsO3 120.0661, obsd 120.0659; b I % D = -31.5' (c = 1.3, EtOH) [lit>2 [a]=D = +88.9' (R,e = 1.36, EtOH). 4-(Hydroxymethyl)butyrolactone (17). The reaction of 4-hydroxy-hxopentanoicacid(2,162mg, 1.42mmol) withDHAP in D2O (about 20 mL) was run es described in the condensation procedure to 40% conversion as determined by lH NMR whereupon sodium borohydride (111mg, 3.0 mmol) was added. After the pH was adjusted to 1.0 with 1 N HC1, the reaction mixture was continuously extracted with ethyl acetate (200 mL) for 36 h. The ethyl acetate was concentrated at aspirator pressure and the resulting residue purified by silica gel chromatography (ethyl acetate/hexane (1:1to 51)) to provide 4-(hydroxymethy1)butyrolactone (17,34 mg, 0.29 mmol): lH NMR (CDCb, 400 MHz) 6 4.59 (tdd, J = 7.4,4.5, 2.9 Hz, 1H), 3.85 (dd, J = 12.5, 2.8 Hz, 1H), 3.64 (dd, J = 12.5, 4.5 Hz, 1H), 3.04 (e, 1H), 2.59 (ddd, J = 17.9,10.0,5.9Hz, 1H),2.50 (ddd, J = 17.9,9.7,8.1 Hz, 1 H), 2.23 (dddd, J = 18.9, 9.8, 7.6, 5.9 Hz, 1HI, 2.12 (dddd, J = 18.7,10.0,8.1,6.8 Hz, 1H); 13CNMR (CDCl3,100 MHz) 177.87, 80.85,63.96,28.62,23.07 ppm; HRMS-CI [M + NHd+ calcd for C&Oa 134.0817, obsd 134.0805; [a]% +19.2' (C = 1.8,EtOH) [lit.= [ a ] %=~+31.5' (s, c = 2.66, EtOH)]. 6-(Hydroxymethyl)caprolactone(18). Methyl 5-hYdrOXy6-hexanoate dimethyl acetal (416 mg, 2.0 "01) in water (7.0 mL) was mixed with an aqueous solution of 1N NaOH (3.0 mL). After 45 min the solution was acidified with Dowex 50-X-8ionexchange resin (H+form, 4.0 g). After being stirred for 16 h, the solutionwas fiitered, partially concentrated at aspirator pressure, and neutralized with 1 N NaOH. DHAP (20 mL of a 0.1 M solution, 2 mmol) was added and the solution neutralized with 1 N NaOH. Aldolase was added (50 U),and the reaction was followed by enzymatic assay of DHAP concentration. At approximately 40% consumption of DHAP (2 h), the reaction was quenched with NaBHd (52 mg,1.4 mmol). After 1 h, the solution was acidified with concentrated HCl to pH 1.0 and partially Concentrated at 1Torr. The aqueous solution (30 mL) was then continuously extracted with ethyl acetate (150 mL). After 18 h, the organic layer was dried (MgSOt), filtered, concentrated at aspirator pressure, and purified by silica gel chromatography (eluent: methanoVCHzCl2 (1:lO)) to provide 6-(hydroxymethyl)caprolactone (12 mg) and ethyl 5,6-dihydroxyhexanoate. This ethyl ester, Dowex 50-X-8 ion-exchange resin (H+form, 0.5 g), and acetonitrile (40 mL) were stirred together. After 16 h, the solution was filtered, concentrated at aspirator pressure, and purified by silica gel chromatography (eluent: CH3CN/CH2C12 ( 1 : l ) ) t o p r o v i d e 1 4 . 4 mg of 6-(hydroxymethyl)caprolactone:'H NMR (400 MHz, CDCb) 6 4.40 (ddt, J = 11.3, 5.6,3.3 Hz, 1H), 3.78 (dd, J = 12.3,3.2 Hz, 1H), 3.65 (dd, J = 12.3, 5.6 Hz, 1H), 2.61 (dddd, J = 17.7,6.5, 4.9,1.4 Hz, 1H), 2.45 (ddd, J = 17.7,9.3,7.0 Hz, 1H), 2.0-1.81 (m, 4 H (OH)), 1.75-1.65 (m, 1H); 13CNMR (100 MHz, CDCb) 171.43, 81.04, 64.87, 29.56, 23.59, 18.33 ppm; HRMS-CI [M + H]+ calcd for C6HloOs 131.0708, obsd 131.0699; [@D = +25.92' 1.2, CHCls) [lit.% [alZD = +34.68' (s, c = 1.3, CHCb)]. (C Methyl 7,8-Dihydroxyoctanoate (19). Methyl 7-hydroxy&hexanoak dimethyl acetal (387 mg, 1.65 mmol) in water (8.0 mL) was mixed with an aqueous solution of 1N NaOH (2.2 mL). After 2 h, the solution was lyophilized, dissolved in 150 mL of H20, and acidified with Dowex 50-X-8 ion-exchange resin (H+ form, 4.0 g). After being stirred for 48 h, the solution was neutralized with 1 N NaOH to pH 6.3, filtered, and partially concentrated at aspirator pressure to 15 mL. DHAP (10 mL of a 0.1 M solution, 1mmol) was added and the solution neutralized with 1N NaOH. Aldolase was added (100 U),and the reaction was followed by enzymatic assay of DHAP concentration. At approximately 25% consumption of DHAP (2 h), the reaction was quenched with NaBH, (100 mg, 3.4 mmol). After 1h, the solution was lyophilized, dissolved in CHsOH (50 mL), and acidified with Dowex 50-X-8ion-exchange resin (H+form, 5.0g). After being stirred for 48 h, the solution was filtered through Celite, concentrated at aspirator pressure, and purified by silica gel chromatography (eluent: ethyl acetate/methanol(loO:Ogoing
+
1894 J. Org. Chem., Vol. 58, No. 7, 1993
Lees and Whitesides
hydrogen atmosphere. After 30 min, the solution was filtered through Celite and concentrated at aspirator pressure to provide 208 mg (0.96 mmol,96%) of 7,&O-isopropylidene-7(R),&dihydroxyoctanoic acid lH NMR (CDCh, 400 MHz) 6 4.07-3.98 (m, H-&,H-7), 3.47 (t,J = 7.3 Hz,H-8b), 2.32 (t,J = 7.4 Hz,H-2), 1.65-1.56 (m, 3 H),1.51-1.25(m, 5 H),1.37 (e, 3 H),1.32 (e, 3 H); l3C NMR (CDCh, 100 MHz)179.82,108.68,75.93,69.36,33.87, 33.28,28.98,26.88,25.68,25.39,24.46ppm; HRMS-FAB[M+ HI+calcd 217.1440,obsd 217.1449; [ ~ I = D= +13.6' (c * 1.4). Anal. Calcd for C11Hm04: C, 61.09;H,9.32. Found: C,61.19; H,9.40. Methyl 7(S),8-Dihydroxyoctanoata (19). Dower 50W-X8 ion-exchange resin (H+ form, 100mg),7,&O-isopropylidene-7(R),8 dihydroxyoctanoic acid (45 mg, 0.21 mmol), and methanol (5.0 mL) were stirred for 24 h, fitered, concentrated at aepirator pressure,and purified by silica gel chromatography(eluent: ethyl acetate/methanol (1oO:O going to 201)) to provide 34 mg (0.18 "01, 85%) of methyl 7(S),&dihydroxyoctanoate: lH NMR (CDCh with a drop of D20,400 MHz) 6 3.68-3.60 (m, 1 H),3.63 (e, 3 H),3.57 (dd, J = 11.2,2.8Hz,1 H),3.36 (dd, J = 11.2,7.7 Hz, 1 H),2.27 (t, J = 7.5 Hz,2 H),1.59 (pentet, J = 7.4 Hz,2 H), 1.46-1.28 (m, 6 H);'H NMR (CDCh, 400 MHz) 6 3.68-3.55 (m, 2 H),3.62 (s,3H),3.40-3.34(br m, 1 H),3.05-2.65 (m, 2 H, OH), 2.27 (t,J = 7.5 Hz, 2 H),1.59 (pentet, J = 7.4 Hz,2 H), 1.46-1.28 (m, 6 H);13CNMR (CDCh, 100 MHz) 174.34,72.09, 66.70,51.51,33.88,33.78,28.99,25.12,24.70ppm; HRMS-FAB [M+ HI+d c d 191.1283,obsd 191.12% [CY]=D -0.79' (C 1.78, CHCh). Anal. Calcd for CsH1804: C, 56.82; H,9.54. (dd,J=8.0,6.2Hz,lHcis),3.53(t,J=8.1Hz,lHtrans),3.49 Found C, 56.94;H,9.42. (t, J = 8.0 Hz, 1 H cis), 2.33 (t, J = 7.3 Hz,1 H cis), 2.32 (t, J = 7.2 Hz,1 H trans), 2.21-2.06 (m, 2 H),1.74-1.67 (m, 2 H),1.39 Acknowledgment. We would like to thank Prof. J. (8, 3 H),1.36 (8, 3 H);1% NMR (100MHzCDCls) 179.43 (cis), Sygusch (Universite de Sherbrooke) for kindly providing 134.27 (trans), 133.55 (cis), 128.38 (trans), 128.27 (cis), 109.15 us an updated version of the crystal structure of RAW. (cis), 77.10 (trans), 71.74 (cis), 69.34 (trans), 69.30 (cis), 33.23 (trans), 33.14 (cis), 31.37(trans), 26.84 (cis), 26.71 (trans), 26.65 Supplementary Material Available: Experimental pro(cis), 25.88 (cis), 24.34 (cis), 23.68 (trans) ppm; HRMS-FAB[M cedures and characterization data for the synthesis of aldehydes + HI+calcd for CllH1804 215.1283,obsd 215.1298. 1-7 and characterization data for sugar phosphates 9-12 (16 ~ ~ O I e o p r o p y l i ~ n ~ 7 ( S ) Acid. ~ y ~ Plat. ~ ~ o i cpages). This material is contained in libraries on microfiche, immediately follows this article in the microfilm version of the inum (10% on carbon, 45 mg) and 7,&O-isopropylidene-7(R),& journal, and can be ordered from the ACS; see any current dihydroxyod-5enoic acid (214 mg, 1.0 mmol) were added to masthead page for ordering information. hexane (10mL). The reaction mixture was then put under a
to 201)) to provide 155 mg of methyl 7,&dihydroxyoctanoate. Spectral properties were identical to those of methyl 7(S),& dihydroxyoctanoate (vide infra) with the exception of the magnitude of the optical rotation: [C7]=D = -0.73' (c = 1.81). 7,8-OIsopropylidene-7(S),8-dihydroxyoct-S-enoicAcid (21). (4-Carboxybuty1)triphenylphosphonium bromide (1.356 , g, 3.0 mmol) in dry THF (80mL) was sonicated for 10 min. Potassium bis(trimethylsily1)amide (12mL of a 0.5 M solution was then added over a 10-minperiod. After in toluene, 6.0"01) 45min, the reaction mixture waa cooled to -20'C, and R-solketal (20,392mg, 3.0 mmol) in THF (5mL) waa added over a 10-min period. After 30 min, the solution was warmed to 21 "C. After 1 h the solution was partially concentrated at aspirator pressure and partitioned between ether (200mL) and pH 3.0 water (0.1 M phosphate). The organic phase was dried (MgSO4), filtered, concentrated at aspirator pressure, and purified by silica gel chromatography (eluent: hexane/ethyl acetate/acetic acid (3: 1:O going to 5:1:0.25)) to provide 446 mg (2.1 mmol, 70%) of 7,&O-isopropylidene-7(R),&dihydroxyod-5-enoic acid (91mixture cis/trans alkene). Only the distinct proton resonances correspondingto the trans alkene are reported. The other proton resonances corresponding to the trans alkene are presumed to occur at approximately the same chemical shift as the corresponding proton resonances in the cis compound: 'H NMR (400 MHz, CDCL) 6 5.73 (dt, J = 15.3,6.8 Hz,1 H trans), 5.58 (dt, J = 10.9,7.5 Hz, 1 H cis), 5.44 (br t, J = 9.8Hz,1 H cis), 4.79 (br q,J = 7.6 Hz,1 H cis), 4.44 (br q,J = 7.3Hz,1 H trans), 4.04
